Antibody detection of antibodies to viral proteins in serum

ABSTRACT

Protein compositions containing the p18 and p25 proteins of the lymphadenopathy virus are used for detecting antibodies in blood serum as indicative of infection by such virus. The proteins can be used in various conventional ways to perform immunoassays for the detection of the antibodies.

This application is a continuation of application Ser. Nos. 07/159,430,filed Feb. 17, 1988, which is a continuation of U.S. Ser. No.06/914,198, filed Oct. 1, 1986, which is a continuation of U.S. Ser. No.06/712,959, filed Mar. 18, 1985, which is a continuation-in-part of U.S.Ser. No. 06/558,109, filed Dec. 5, 1983 all now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

Retroviruses as the vectors of human diseases have only recently beenestablished. The first retrovirus shown to cause cancer was the humanT-lymphoma/leukemia virus-I (HTLV-I). Poiosz et al., Proc. Nat'l. Acad.Sci. (1980), 77, 6815. A second retrovirus was reported as being presentin hairy cell leukemia cells and was referred to as HTLV-II.Kalyanaraman et al., ibid. (1982) 218:571. These two viruses are capableof transformation, have a T-cell tropism and a reverse transcriptaserequirement for magnesium.

With lymphadenopathy syndrome (LAS) or acquired immune deficiencysyndrome (AIDS) becoming a major health threat, substantial efforts havebeen made to determine the cause of the disease. The syndrome differedfrom the diseases caused by the human retroviruses HTLV-I and HTLV-II,in that the agent did not transform the host cells to a neoplasticstate, but rather was cytotoxic to the host cells. It shared a similarproperty to the earlier discovered retroviruses in having a T-celltropism, particularly helper cells. It was early found that the syndromecould be transmitted by blood transfusions. A carrier of the disease whowas a source of blood could be a transmitter of the disease to a personreceiving that blood. It has, therefore, become a major effort to findways to screen blood to detect whether the donor may have been infectedwith the pathogenic cause of the disease.

2. Description of the Art

Descriptions of LAV, HTLV-III and ARV may be found in Barre-Sinoussi etal., Science (1983) 220:868; Montagnier et al., Cold Spring HarborSymposium (1984), in press; Vilmer et al., Lancet (1984) 1:753; Popovicet al., Science (1984) 224:497; Gallo et al., Science (1984) 224:500;Feorino et al., Science (1984) 225:69-72; Klatzman et al., Science(1984) 225:59-62; Montagnier et al., ibid. (1984) 225:63-66; Levy etal., Science (1984) 225:840-842; Montagnier et al., Ann. Virol.(Institut Pasteur 1984, 135E, 119-134; Kalyanaraman et al., Science(1984) 225:321; and Ray et al., Science (1984) 225:321; and Ray et al.,Biochenm. and Biophys. Res. Comm. (1984) 121:126.

SUMMARY OF THE INVENTION

Methods and compositions are provided for detecting the presence ofantibodies to proteins associated with lymphadenopathy virus (LAV).Particularly, the compositions contain at least one of the p18 or p25proteins or immunologically active fragments thereof, and preferablycontain both the p18 and p25 proteins, which are used in a reagent,either labeled or unlabeled, for the detection of serum antibodies toLAV or other immunogenically proteins as indicative of infection by LAVor related viruses.

DESCRIPTION OF SPECIFIC EMBODIMENTS

Methods and compositions are provided for detection of the probableincidence of infection with a T-cell tropic virus, whose infection inhumans results in substantial diminution in the helper/suppressor T-cellratio. The T-cell tropic retrovirus is characterized by having a densityin a sucrose gradient of 1.16. The reverse transcriptase enzyme has amagnesium requirement with an optimal concentration of about 5 mM and anoptimal pH of about 7.8. The reaction is not inhibited by actinomycin D.The reverse transcriptase activity displays a strong affinity forpoly(adenylate-oligodeoxythymidylate).

Electron microscopy of ultrathin sections of virus-producing cells showstwo types of particles, corresponding to the immature and mature formsof the virus. Immature particles bud at the cell surface with a densecrescent in close contact with the plasma membrane. Occasionally, someparticles remain in this state, while being freed from the cell surface.Mature particles have a different morphology with a small, dense,eccentric core, mean diameter: 41 nm. Most of the virions are round(mean diameter: 139 nm) or ovoid, but in some instances, a tailedmorphology can be observed. This latter form can be observed incytoplasmic vesicles which are released in the media. Such particles arealso formed by budding from vesicle membranes.

The retrovirus is tropic for helper T-lymphocytes (OKT4 cells). Thesecells are characterized in part by binding to monoclonal antibodies,such as those designated OKT4 (Ortho).

In most cases of the lymphotropic disease, the ratio of OKT4 cells toOKT8 cells, which is normally greater than 1, is depressed to valuesbelow 1, being 0.1 or less. More importantly, the absolute number ofOKT4 cells per mm³ of blood is severely reduced in these patients.

The subject retroviruses are immunologically distinct from thepreviously known HTLV-I. A monoclonal antibody to p19 of HTLV-I(Robert-Guroff et al., J. Exp. Med. (1981) 154:1957) and a polyclonalgoat antibody to p24 of HTLV-I (Kalyanaraman et al., J. Virol (1981)38:906) were used in an indirect fluorescence assay. Infected cells fromthe biopsy of a patient having the subject virus and lymphocytesobtained from a healthy donor and infected with the same virus wereassayed. The virus-producing cells did not react with either antibody,whereas two lines of cord lymphocytes chronically infected with HTLV-I(Popovitch et al., Science (1983) 219:856) and used as controls showedstrong surface fluorescence.

In addition, proteins detected on polyacrylamide gel electrophoresis ofa lysate of cells infected with the subject virus included p13, p18,p25, p36, p42 and p80 proteins (the numbers indicate the approximatemolecular weight in kilodaltons). The retroviruses of the subjectinvention are, therefore, those viruses producing the same or similarproteins where said same or similar proteins are substantiallycompletely immunologically cross-reactive which said proteins detectedin said lysate, particularly as to epitopic sites specific for p18 andp25 of LAV.

The subject method is therefore applicable to detection of infection inhumans as a result of viruses having all or substantially all of theabove characteristics and producing proteins cross-reactive with the p18and p25 proteins observed with the subject LAV virus.

The compositions which are employed in the assays of the subjectinvention will have one or both, preferably both, the p18 and p25proteins or immunologically active fragments thereof, either natually orsynthetically derived, having epitopic sites specific for LAV. The p25and p18 proteins appear to be core proteins. The compositions employedin the subject invention may be derived from a variety of sources,either naturally occurring or synthetic. At least 10% by weight,preferably at least 20% by weight, of the polypeptide present in thecomposition will be related to epitopic sites of the p18 and/or p25proteins. Up to about 100% by weight of the polypeptides in thecomposition can be related to p18 or p25 proteins or mixtures thereof.Preferably, polypeptides related to both p18 and p25 proteins will bepresent in the composition in an amount of at least about 15 percent byweight and up to about 100% by weight. Frequently, polypeptides relatedto both p18 and p25 proteins will not be more than about 65% by weight.Amounts of the p18 or p25 proteins or mixtures thereof may be employedin the lower portion of the range where the other proteins present inthe protein. composition are derived from or related to epitopic sitesassociated with the subject virus.

The retroviral composition may be purified by any conventionaltechnique, e.g., density gradient separation. The polypeptidecompositions employed in the assay may come from a lysate prepared bytreating a substantially purified viral composition with a detergent orother chaotropic agent to disaggregate the proteins. The resultinglysate may be further purified by chromatography, e.g., affinitychromatography, electrophoresis, or the like. Individual components fromthe lysate, when separated, can be recombined to provide forsubstantially the same or different proportions of the differentpolypeptide components. The compositions will be at least substantiallyfree of polypeptides resulting from expression of structural genes ofthe human genome, where the expression is unrelated to the retroviralinfection.

Alternatively, all or portions of the viral RNA may be reversetranscribed to provide single-stranded DNA (ss DNA), which can be usedas a template to provide double-stranded DNA (ds DNA). The ds DNA can beintroduced into an appropriate mammalian vector for introduction into acompatible host for expression of some or all of the viral proteins.Alternatively, one or more structural genes of the virus may beidentified, ds DNA prepared from the RNA encoding for such structuralgene and introduced into a prokaryotic or eukaryotic expression vectorhaving the appropriate transciptional and translational initiation andtermination signal sequences for expression. For glycoproteins it willusually be desirable to employ a host which provides for appropriateglycosylation, so as to maximize the immunologic similarity of theglycoprotein produced by recombinant techniques, as compared to thenaturally occurring glycoprotein.

The related oligopeptides having epitopic sites of p18 and/or p25 (whichmay be on the same or different molecules) will be of at least 12 aminoacids, more usually at least 18 amino acids, and may be 30 amino acidsor more, up to and exceeding the number of amino acids of the parentpolypeptide. These oligopeptides may be synthesized chemically, bycloning, or as fragments of larger polypeptide precursors. Theoligopeptides may be joined to longer proteins in some instances.

The subject protein compositions can be employed in any of a variety ofconventional immunoassays. These assays employ a wide variety of labelsand provide for a varying range of sensitivity and susceptibility tointerference. Labels include radionuclides, enzymes, fluorescers,chemiluminescers, particles, ligands, enzyme substrates, enzymecofactors, enzyme inhibitors, particularly suicide inhibitors, lightemitter-quencher combinations, etc. The immunoassays may be homogeneousor heterogeneous, where the distinction relates to the use of aseparation step for separating uncomplexed label from complexed label.

Depending upon the protocol, the protein composition of the subjectinvention may be labeled or unlabeled. For example, the p18-p25 proteincomposition may be covalently or non-covalently bound to the surface ofa microtiter plate well. A serum sample suspected of containing antibodymay then be added, unbound antibody washed away and either labeledanti-human IgG or labeled p18-p25 composiiton added. The advantage ofthe latter is that it provides for a lower amount of non-specificbinding of the label. Thus, either the anti-human IgG or the p18-p25protein composition may be labeled with radionuclides, enzymes, or thelike and the signal detected in accordance with conventional techniques.Various radioisotopes include ³⁵ S, 32_(P), 125_(I), 3_(H) 152_(Eu),etc. For enzymes, a variety of enzymes can be used, such as horseradishperoxidase, glucose-6-phosphate dehydrogenase, glucose oxidase, alkalinephosphatase, β-galactosidase and the like. A wide variety of substratesare available which allow for colorimetric or fluorimetric detection.For fluorescers, fluorescein, dansyl, rhodamine, Texas red,phycobiliproteins, or the like may be employed. The choice of the assaymethod will primarily depend upon the sensitivity desired, theconvenience of the assay, and the availability of the reagents.

Conveniently, kits can be provided having the subject proteincompositions in conjunction with the appropriate reagents. Theappropriate reagents may include labeled protein compositions, labeledanti-human immunoglobulin, or labeled anti-p18 and/or anti-p25. Inaddition, as a control, a lymphocyte extract obtained from a healthyperson may be provided, as well as serum from a healthy person forstandards. In addition, buffers, stabilizers, or other ancillaryadditives may also be included. The materials will normally be providedas lyophilized compositions, which can be reconstituted to the desiredconcentrations. In addition, depending upon the particular protocol,other materials may be provided, such as enzyme substrates, cofactors,or the like.

Where reagents are combined, they will normally be in proportion to atleast substantially maximize the sensitivity of the assay. Where theactive reagents are in separate containers, the proportions will be inpart a matter of convenience, since each will be reconstituted in aprescribed volume to be in appropriate relative proportion with thevolumes of the other reagents.

The following examples are offered by way of illustration and not by wayof limitation.

EXPERIMENTAL 1. Virus Propagation

Cultured T-lymphocytes from either umbilical cord peripheral blood, orbone marrow cells from healthy, virus negative, adult donors aresuitable for virus propagation.

There is, however, some variation from individual to individual in thecapacity of lymphocytes to grow the virus. Therefore, it is preferableto select an adult healthy donor, who had no antibodies against thevirus, as detected by reverse transcriptase activity (RT), nor expressedviral proteins.

Lymphocytes of the donor were obtained and separated by cytophoresis andstored until used frozen at -180° C. in liquid nitrogen, in RPMI 1640medium, supplemented with 50% decomplemented human serum and 10% DMSO.

For viral infection, lymphocytes were put in culture (RPMI 1640 medium)with phytohaemagglutinin (PHA) at a concentration of 5×10⁶ cells/ml forthree days. The medium was then removed and cells resuspended in viralsuspension (crude supernatant of virus-producing lymphocytes, stored at-80° C.). Optimal conditions of cell/virus concentrations were 2×10⁶cells for 5 to 10,000 cpm of RT activity, the latter determined asfollows.

The cell-free supernantant was ultra-centrifuged for 1 hr at 50,000rev/min. The pellet was resuspended in 200 ul of NTE buffer (10 mM Tris,pH 7.4, 100 mM NaCl, and 1 mM EDTA) and was centrifuged over a 3 mllinear sucrose gradient (10 to 60 percent) at 55,000 rev/min for 90 minin an IEC type SB 498 rotor. Fractions (200 ul) were collected, and 30ul samples of each fraction were assayed for DNA RNA-dependentpolymerase activity with 5 mM Mg²⁺ and poly(a)-oligo-(dT)₁₂₋₁₈ astemplate primer. A 20 ul portion of each fraction was precipitated with10% trichloroacetic acid and then filtered on a 0.45 um Milliporefilter. The ³ H-labeled acid precipitable material was measured in aPackard counter.

After 24 hr, cells were centrifuged to remove the unadsorbed virus andresuspended in culture PHA-free medium and supplemented with PHA-freeTCGF (Interleukin 2): (0.5-1 U/ml, final concentration), Polybrene(Sigma) 2 ug/ml and sheep anti-interferon serum, inactivated at 56° C.for 30 min (0.1% of a serum which is able to neutralize 7 U of αleukocyte interferon at a 1/100,000 dilution).

Virus production was tested every 3 days by an RT activity determinationon 1 ml samples.

The presence of anti-interferon serum is important in virus production.When lymphocytes were infected in the absence of anti-human-α-interferonserum, virus production, as assayed by RT activity, was very low ordelayed. The sheep antiserum used was raised against partly purified αleukocyte interferon, made according to the Cantell technique.

Virus production starts usually from day 9 to 15 after infection, andlasts for 10-15 days. In no case was the emergence of a continuouspermanent line observed.

2. VIRUS PURIFICATION

For its use in ELISA, the virus was concentrated by 10%polyethyleneglycol (PEG 6000) precipitation and banded twice toequilibrium in a 20-60% sucrose gradient. The viral band at density 1.16was then recovered and used as such for ELISA assays.

For use in a radioimmunoprecipitation assay (RIPA), purification inisotonic gradients of METRIZAMIDE/or NICODENZ (trademarks of Nyegaard,Oslo) were found to be preferable. Viral density in such gradients wasvery low (1.10-1.11).

Metabolic labeling with radioactively labeled amino acid ³⁵ S-methionineof cord lymphocyte cells and virus was carried out for 20 hr, the cellslysed with detergent, virus banded as described above, lysed with RIPAbuffer in a medium deprived of the amino acid used for labeling,immunoprecipitated, followed by polyacrylamide gel electrophoresis,except the following modifications for RIPA: virus purified in Nycodenzwas lysed in 4 volumes of RIPA buffer (0.5% SDS) containing 500 U/ml ofaprotinin. Incubation with 5 ul of serum to be tested was made for 1 hrat 37° C. and then 18 hr at 4° C. Further incubation of theimmunocomplexes with protein A Sepharose beads was for 3 hr at 4° C.

3. PREPARATION OF THE VIRUS EXTRACT FOR ELISA ASSAYS

Virus purified in a sucrose gradient, as above described, is lysed inRIPA buffer (0.5% SDS) and coated on wells of microtest plates (Nunc).

Preferred conditions for the ELISA assay are described hereafter.

After addition to duplicate wells of serial dilutions of each serum tobe tested, the specifically fixed IgGs are revealed by goat anti-humanIgG coupled with horseradish peroxidase. The enzymatic reaction iscarried out on ortho-phenylenediamine (OPD) as substrate and read withan automatic spectrophotometer at 492 nm.

On the same plate each serum is tested on a control antigen (a crudecytoplasmic lysate of uninfected T-lymphocytes from the same donor) inorder to eliminate non-specific binding, which can be high with somesera.

Sera are considered as positive (possessing antibodies against thevirus) when the difference between O.D. against the viral antigen andO.D. against control cellular antigen was at least 0.30.

Method

This ELISA test is for detecting and titrating seric anti-retrovirustype LAV antibodies. It comprises carrying out a competition testbetween a viral antigen (cultivated on T-lymphocytes) and a controlantigen constituted by a lysate of the same though non-infectedlymphocytes.

The binding of the antibodies to the two antigens is revealed by the useof a human antiglobulin labeled with an enzyme which provides adetectable signal in conjunction with a suitable substrate.

Preparation of the viral antigen

The cell cultures which are used are T-lymphocytes of human origin whichcome from:

umbilical cord blood,

bone marrow,

blood of a healthy donor.

After infection of the cells by the virus, the supernatant of theinfected cell culture is used. It is concentrated by precipitating with10% PEG, then purified (two or three times) on a (20-60%) sucrosegradient by ultracentrifugation to equilibrium.

The viral fractions are gathered and concentrated by centrifugation at50,000 rev/min for 60 min.

The sedimented virus is taken up in a minimum volume of NTE buffer (Tris0.01M (pH 7.4), NaCl 0.1M, EDTA 0.001M). The protein concentration isdetermined by the Lowry method. The virus is then lysed by a (RIPA+SDS)buffer (0.5% final) for 15 min at 37° C.

Preparation of the control antigen

The non-infected lymphocytes are cultured according to the precedingconditions for from 5 to 10 days. They are centrifuged at low speed andlysed in the RIPA buffer in the presence of 5% of Zymofren (Specia) (500μ/ml). After a stay of 15 min at 4° C. with frequent stirrings with avortex, the lysate is centrifuged at 10,000 rev/min. The supernatantconstitutes the control antigen. The protein concentration is measuredby the Lowry method.

Reagents

1-Plates=NUNC-special controlled ELISA

2-Buffer PBS: pH 7.5

3-TWEEN 20

4-Carbonate buffer: pH=9.6 (CO₃ Na₂ =0.2M) (CO₃ HNa=0.2M)

5-Non-fetal calf serum: which is stored in frozen state (BIOPRO),

6-Bovine serum albumin (BSA) (SIGMA fraction V)

7-Human anti IgG (H+L) labeled with peroxidase (PASTEUR), in 1 ml tubespreserved at 4° C.

8-Washing buffer=PBS buffer, pH 7.5+0.05% TWEEN 20 Dilution of theconjugate is carried out at the dilution indicated in PBS buffer+TWEEN20 (0.5%)+(BSA), 0.5 g/100 ml

9-Dilution buffer of sera=PBS buffer+0.05% TWEEN 20+0.5 g BSA per 100 ml

10-Substrate=OPD

Citrate buffer pH=5.6 trisodio citrate (C₆ H₅ Na₃ O₇, 2H₂ O), 0.05M;citric acid (C₆ H₈ O₇, 1H₂ O), 0.05M.

Hydrogen peroxide=at 30% (110 volumes)used at 0.03% when using citratebuffer.

OPD-(SIGMA)=75 mg per 25 ml of buffer (freshly prepared).

Preparation of the plates

The plates which are used have 96 U-shaped wells (NUNC).

The distribution of antigens is as follows:

100 μl of the viral antigen diluted in carbonate buffer at pH 9.6, aredeposited in each of the wells of rows 1-2-5-6-9-10

100 μl of the control antigen, diluted in carbonate buffer at pH 9.6,are deposited in each of the wells of rows 3-4-7-8-11-12.

The dilution of the viral antigen is titrated with each viral product.Several dilutions of viral antigen are tested and compared to positiveand negative known controls (at several dilutions) and to human anti-IgGlabeled with peroxidase, the latter also being tested at severaldilutions.

As a rule, the protein concentration of the preparation is from 5 to 2.5μg/ml. The same protein concentration is used for the control antigen.

The plates are covered with a plastic lid and are incubated overnight at4° C.

They are then put once in distilled water and centrifuged. The wells arethen filled with 300 μl of non-fetal calf serum at 20% in PBS buffer andincubated for 2 hr at 37° C. (covered plates). The plates are washed 3times in PBS buffer with TWEEN 20, 0.5% (PBS-TWEEN buffer):

first washing 300 μl

second and third washing 200 μl/well.

The plates are carefully dried and sealed with an adhesive plastic film.They can be stored at -80° C.

ELISA reaction: antibody titer assay

After defrosting, the plates are washed 3 times in PBS-TWEEN. They arecarefully dried.

The positive and negative control sera as well as the tested sera arediluted first in the tube, with PBS-TWEEN containing 0.5% BSA.

The chosen dilution is 1/40.

100 μl of each serum are deposited in duplicate on the viral antigen andin duplicate on the control antigen.

The same is carried out for the positive and negative diluted sera.

100 μl of PBS+TWEEN+BSA are introduced in two wells (viral antigen) andin two wells (control antigen) to form the conjugated controls.

The covered plates are incubated for 90 min at 37° C.

They are washed 4 times in PBS-TWEEN.

100 μl of human anti-IgG (labeled with peroxidase) at the choosendilution are added to each well and incubated at 37° C.

The plates are again washed 5 times with PBS+TWEEN buffer and carefullydried.

The enzyme reaction is carried out with 100 μl OPD substrate (0.05% incitrate buffer pH 5.6 containing 0.03% of H₂ O₂) in each well. Theplates are left in a dark room for 20 min at room temperature. Readingis carried out on a spectrophotometer (for microplates) at 492 nm.

Sera deemed as containing antibodies against the virus are those whichgive an ODD (optical density difference=optical density of viral antigenless optical density of control antigen) equal to or higher than 0.30.

This technique enables a qualitative titration as well as a quantitativeone. For this purpose, it is possible either to use several dilutions ofthe serum to be assayed or to compare a dilution of the serum with arange of controls tested under the same conditions.

The following table provides the results of serological investigationsfor LAV antibodies carried out by using the above exemplified ELISAassay.

                  TABLE                                                           ______________________________________                                        Results of Serological Investigations for LAV Antibodies                                                 ELISA-HTLV1.sup.+                                  Total        ELISA-LAV     (Biotech)                                          examined     positive/% positive                                                                         positive/% positive                                ______________________________________                                        Lympha- 35       22       (63)    5**   (14)                                  denopathy                                                                     patients*                                                                     Healthy 40       7        (17)   1       (3)                                  homo-                                                                         sexuals                                                                       Control 54       1          (1.9)                                                                              0      (<2.6)                                population                                                                    ______________________________________                                         *28 homosexuals, 3 Haitians (1 woman), 4 toxicomans (2 women)                 .sup.+ The number of positive sera is probably overestimated in this test     since no control of nonspecific binding could be done.                        **Out of the 5 LAS HTLV1 positive, 3 were born in Haiti, 1 had stayed for     a long time in Haiti and 1 had made several travels to USA. All of them       also had antibodies against LAV.                                         

The table shows clearly high prevalence of LAV antibodies in thehomosexual patients with LAS, a very low incidence in the normalpopulation and also a moderate spread of virus infection in stillhealthy homosexuals. In the latter group, all the positive individualshad a high number of partners (>50 per year). The groups of AIDSpatients gave less interpretable results: approximately 20% had LAVantibodies, but some of the sera were taken at a very late stage of thedisease, with a possible inhibition of the humoral response.

It should be further mentioned that lymphocytes of all LAS patients donot produce detectable amounts of LAV-type virus. Particularly cells oflymph nodes from 6 more LAS patients were put in culture and tested forvirus production. No virus release could be detected by RT activity.However, a p25 protein recognized by the serum of the first patientcould be detected in cytoplasmic extracts of the T-cells labeled with ³⁵S-methionine in 3 other cases. This suggests partial expression of asimilar virus in such cases. Moreover, all (6/6) of these patients hadantibodies against LAV p25 protein, indicating that they all had beeninfected with a similar or identical virus.

The LAV1 virus has been deposited at the "Collection Nationale desCultures de Micro-organismes" (C.N.C.M.) 28 rue du Docteur Roux, 75724Paris Cedex 15, under No. I-232 on Jul. 15, 1983 and IDAV1 and IDAV2viruses have been deposited at the C.N.C.M. on Sep. 15, 1983 under No.I-240 and I-241, respectively. The invention encompasses as well theextracts of mutants or variants of the above deposited strains as longas they possess substantially the same immunological properties.

A competitive RIA of the p25 protein of LAV and analogous core proteinsof other retroviruses was carried out. The RIAs were carried out with¹²⁵ I-labeled LAV p25 and limiting dilution of human serum positive forantibodies to LAV (B.R.U. patient). Serial dilutions (100 μl) ofsolubilized virus in buffer 1' (20 mM disodio acid phosphate (pH 7.6), 2mM NaCl, 1 mM EDTA, 0.3% Triton X-100, 0.1 mM phenylmethylsulfonylfluoride, and bovine serum albumin (2 mg/ml) were incubated with theappropriate serum for 1 hr at 37° C. Labeled LAV p25 (8000 counts/min in50 μl of buffer 1) was then added, and the mixture was further incubatedat 37° C. and at 4° C. overnight. A 20-fold excess of goat antiserum tohuman IgG was then added and the volume made up to 1 ml in buffer 1. Thesamples were further incubated at 37° C. for 1 hr and at 4° C. for 2 hrand then centrifuged at 2500 rev/min for 5 min. The supernatant fluidswere aspirated and the radioactivity in the sediment was determined in agamma counter. It was found that unlabeled p25 from LAV competed wellfor homologous antibody (from patient B.R.U.) while other retrovirusesdid not. The non-competing viruses included HTLV-I, HTLV-II,Mason-Pfizer monkey virus, Simian sarcoma virus, baboon endogenousvirus, Rauscher murine leukemia virus, mouse mammary tumor virus andequine infectious anemia virus.

Serum from AIDS patients was obtained either as part of various studiessponsored by the Centers for Disease Control (CDC) or as individualspecimens submitted directly to CDC. Serum from LAS patients wasobtained as part of an ongoing perspective study of the syndrome amonghomosexual men in Atlanta. Serum from CDC employees who worked inlaboratories in other areas was obtained in 1983 and 1984; some of thelaboratory workers were exposed to materials from AIDS patients. Serumsamples were also collected from homosexual men, 18 years of age orolder, who sought medical care at the San Francisco City Clinic. Thesesamples were routinely collected from 1978 as part of ongoing studies ofHepatitis B virus infection in homosexual men; specimens from 1984 wereobtained according to the same protocol as part of a Hepatitis B vaccinestudy. Serum from blood donors (provided by the National Red Cross) waschosen at random from samples obtained in 1980 and 1981.

Specific antibody to the core protein of LAV p25 was present in serumfrom 41% of the AIDS patients and from 72% of the LAS patients. Noantibody was detected in serum from CDC employees or from the blooddonors.

The prevalence of antibodies to LAV p25 varied only slightly amongdifferent groups of AIDS patients. There was considerable variation,however, among AIDS patients with different disease manifestations.Patients with Kaposi's sarcoma alone had a significantly higher antibodyprevalence [22 (63%) of 35] compared with patients who had onlyopportunistic infections [29 (34%) of 90] (P=<0.005, Students t-test).These results are consistent with the observation that Kaposi's sarcomapatients are less immunodeficient that patients with opportunisticinfections.

Antibodies to p18 have been detected in the sera of a majority oflymphadenopathy and AIDS patients by Western blotting andradioimmunoprecipitation assay. In general, Western blotting appears tobe a more sensitive means of detecting antibody to p18 than isimmunoprecipitation. Antibody to p18 also appears to be more prominentearly in the disease course than late.

In accordance with the subject invention, compositions are providedwhich can be used in a wide variety of methods for screening serum forthe presence of antibodies to the subject retrovirus. Thus, the subjectcompositions provide for protection against the presence of theretrovirus in serum for transfusion, in indicating the presence or priorexistence of infection of the subject virus, and in providing means fordistinguishing among the various T-cell tropic viruses.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

What is claimed is:
 1. A diagnostic method for detecting the presence ofantibodies that specifically bind to antigens of human immunodeficiencyvirus (HIV), comprising:(a) contacting a radioactively labeled lysate ofHIV-1 comprising p18 antigen with a biological sample for a time andunder conditions sufficient to permit formation of antigen-antibodycomplex between said p18 antigen and said antibodies; and (b) measuringthe formation of said complex to determine the amount of p18 antigenpresent in the sample.
 2. The method according to claim 1, wherein saidbiological sample is from a human.
 3. The method according to claim 2,wherein said amount of p18 antigen present in the sample is used todetermine the amount of p18 antigen present in the human.
 4. The methodaccording to claim 3, further comprising determining the amount of freecomplexing antibody to p18 antigen of HIV present in the human.
 5. Themethod according to claim 1, further comprising(a) contacting aradioactively labeled lysate of HIV-1 comprising p25 antigen with abiological sample for a time and under conditions sufficient to permitformation of antigen-antibody complex between said p25 antigen and saidantibodies; and (b) measuring the formation of said complex to determinethe amount of p25 present in the sample.
 6. The method according toclaim 5, wherein said biological sample is from a human.
 7. The methodaccording to claim 6, wherein said amount of p18 antigen present in thesample is used to determine the amount of p18 antigen present in thehuman, and said amount of p25 antigen present in the sample is used todetermine the amount of p25 antigen present in the human.
 8. The methodaccording to claim 6, wherein said amount of p25 antigen present in thesample is measured as a sum of the amount of p18 antigen and p25 antigenpresent in the sample.
 9. The method according to claim 7, furthercomprising determining the amount of free complexing antibody to p18antigen of HIV present in the human, and determining the amount of freecomplexing antibody to p25 antigen of HIV present in the human.